3.1.1 TatRGD and DNA Binding Analysis:
We performed binding experiments between TatRGD (TR) and DNA, to determine an appropriate concentration ratio of TR and DNA. Firstly, the standard curve was obtained by dotting biotin-TRD (containing 1µg DNA mixed with 2, 4, 6, 12.5 or 25µg TR) directly (with out column separation) on the membrane and backing at 60oC. The dots were developed, quantified and used to obtain the standard curve. The intensity of the dots obtained were directly proportional to the amount of TR employed (figure 9-A).
TatRGD-DNA (TRD) complexes were prepared by mixing 1µg DNA with corresponding concentrations (2, 4, 6, 12.5, 25µg) of biotin-TR in HBS at physiological pH. These TRD mixtures were passed through Sephadex G-100 columns and eluted in 10 fractions containing 6 drops each. When biotin-TR alone was passed through the column no dots were found on the blotting membrane until 12 fractions (no color appeared on the blots).
Hence the TRD complexes were eluted in 10 fractions for each sample. The eluents from Sephadex G-100 seperation were dot-blotted and quantified as mentioned in the methods section. When TRD mixtures were passed through the Sephadex G-100 columns, only stable TRD complexes formed were assumed to be eluted in the first 12 fractions. The results obtained were in correspondence with this. Colored dots were seen only in the 5th and 6th fractions of elution (figure 9-B). By fitting these values in the standard curve the approximate amounts of TR bound per µg DNA was obtained.
The binding curve was then plotted with the input amount of TR on the x-coordinate and the amount of TR bound to 1µg DNA on the y-coordinate (figure 9-C). Figure 9-B and C suggests that, a minimum of 4µg TR is required per µg DNA to obtain TRD complexes.
Binding saturated at approximately 12 µg TR per µg DNA.
6
A
2 4 12.5 25 µg
Figure 9: TRD binding analysis. Dot blots obtained by direct application of TRD complexes were used for obtaining the standard curve (A). Dot blots were obtained by dotting eluents from column separation (B). Binding curve suggested the amount of TR bound /µg DNA (C).
3.1.2 Particle size measurement:
Since the size of the particle is one of the important criterions for its cellular internalization, particle size measurements were performed for TRD and TRDL complexes. When TatRGD and DNA were alone measured, the average diameter was beyond the range of the instrument (i.e.) >6µm. But, when TatRGD was mixed with DNA, the average diameter of the TRD complexes reduced ranging between 1µm and 2.5µm. TRD complexes with lower TR concentrations formed particles with smaller diameters. Further reduction in the average diameter was observed when lipofectamine was added to the TRD mixtures. This suggests that, addition of lipofectamine to TRD, results in further compaction of the particles which aids in better internalization.
Dot 5
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B
2 4 6 12.5 25 µg
Biotin-TR (µg) + 1µg DNA [Column purification]
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C
Figure 10: Particle size measurements. Measurement of particle sizes of different amount of TR added to DNA (TRD) or different amounts of TR added to DNA complexed with Lipofectamin (TRDL). Z. Avg represents the average size.
3.1.3 Dose response:
To investigate the applicability of TatRGD as a transfection agent, the effect of different TR amounts per µg DNA on transfection efficiency was studied employing luciferase reporter gene.
In case of A549 cells, significant transfection efficiencies with TRD vectors were observed (figure11-A). Expression levels were observed to be substantially higher when cells were transfected with TRDL vectors obtained by addition of Lipofectamine to TRD mixtures (figure11-B).
In primary pulmonary artery cells, such as adventitial fibroblasts (FBPA) (figure11-C) and smooth muscle cells (SMCPA) (figure11-D), TR alone was not sufficient for DNA transfection. However, TRDL vectors significantly improved the luciferase expression levels over that of Lipofectamine standards.
Careful observation of the cells before adding lysis buffer revealed lowered cell viability at higher concentrations of TR. This may be attributed to the decline in transfection efficiency at higher concentrations of TR in A549 cell-line (25µg) as well as FBPA and SMCPA primary cells (4µg).
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Figure 11: Dose response in A549, FBPA and SMCPA cells. Dose response in A549 cells towards TRD (A) and TRDL (B) mediated transfections. Dose response in FBPA (C) and SMCPA
(D) primary cells towards TRDL mediated transfections.
3.1.4 Cyto-toxicity assay:
To derive an optimal TR concentration for transfection with minimally compromising cell viability, the cytotoxicitiy associated with different concentrations of TRD and TRDL vectors on the A549 cell-line (figure12-A) and FBPA primary cells (figure12-B) was assessed. Increasing amounts of TR enhanced the toxicity. Also, the toxicity was enhanced by the addition of Lipofectamine by at least 5% when delivering TRDL into the cells. Further more, the cellular response to test compounds followed the order: TRDL >
DL > TRD > D.
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Figure 12: Cyto-toxicity assay. Measurement of cytotoxicity in A549 cells (A) and FBPA cells (B) treated by different concentrations of TR in the presence of a given amount (1µg) of DNA (TRD) or of DNA and Lipofectamin (TRDL).
3.1.5 Improvement in transfection efficiency with TRDL vector systems:
Considering the curves of Figures 11 and 12 the optimal concentrations of TR (per microgram of DNA) to be employed in all further analysis using TRDL vectors was estimated as 12.5µg for A549 and 4 µg for FBPA and SMCPA.
The analysis of DNA transfection by TRD, LD, and TRDL was shown in comparison, by measuring the reporter gene luciferase expression in cellular lysate and by counting the GFP positive cells by fluorescence microscopy (figure13).
In A549 cells the transfection efficiencies of TRDL complexes showed 5-fold improvement over the standard lipofectamine (DL) aided transfection. Further, the addition of lipofectamine improved the transfection efficiency of TRD complexes by approximately 30-fold.
In FBPA and SMCPA cells the transfection efficiencies of TRDL vectors showed 3 and 2-fold improvement respectively, over that of DL.
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Figure 13: TRDL improved transfection efficiency. Summary on transgene expression levels employing different mixtures of TRD, DL and TRDL as measured by luciferase reporter gene expression in case of pgl3CMV transfection (RLU: relative light units) (left) or by counting number of fluorescent stained cells in case of pEGFP-N2 (right) transfection in A549 (A), FBPA
(B) and SMCPA (C) cells.
3.1.6 Analysis of DNA uptake:
For characterization of the facilitated gene transfer, the cellular uptake of fluorescent Fam labeled DNA fragments was studied in A549 and FBPA cells transfected with either DL or TRDL. A 409bp fluorescent DNA fragment (Fam-CMV) amplified from pgl3CMV (figure 14-A) was employed for this purpose. The quantity and integrity of the 409bp fragment was analyzed by capillary gel electrophoresis (Gene Scan) (figure 14-B).
Recovering the DNA fragment from the cellular and nuclear compartments of the cells transfected with TRDL and DL vectors exhibited that, TRDL significantly enhanced cellular DNA uptake over that of DL vector system and that the effect is more pronounced in A549 cells (figure 14-C) than in FB (figure 14-D).
Figure 14: Analysis of DNA uptake. Fam-CMV is a 409bp fluorescent DNA fragment amplified from pgl3CMV (A) [Lane 1: PCR amplified product from plasmid. The 409bp fragment was excised and gel eluted. Lane 2: PCR product after gel elution. M: Marker]. The quality and integrity of the DNA fragment can be observed from the electropherogram (B).
Analysis of DNA uptake revealed higher localization of the fluorescent DNA fragment in the cellular and nuclear compartments with TRDL mediated transfection over that of DL mediated transfection, in both A549 cell-line (C) and FBPA primary cells (D). [C: cellular and N: nuclear compartments]
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3.1.7 TRDL transfection efficiency over commercial standards:
Comparison of TRDL mediated transfection with commercially available magnetic beads (CombiMag, Chemicell), Cationic polymer (25 kDa PEI, Aldrich) and cationic lipid (Lipofectamine 2000, Invitrogen) revealed approximately 3.5, 90, 5-fold improvement over CombiMAG, PEI and Lipofectamine 2000 respectively in A549 cells (figure 15-A), 1.3, 23, 3-fold respectively in FBPA cells (figure 15-B) and 4, 65, 2-fold respectively in SMCPA cells (figure 15-C).
Figure 15: TRDL transfection efficiency over commercial standards. TRDL mediated transfection was observed to be superior over other commercially available transfection agents in A549 cell-line (A) and FBPA (B), SMCPA (C) primary cells.
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3.1.8 Immunostaining and confocal imaging:
Caveoli mediated internalization of TRDL complexes:
To explore if the mode of internalization exploited by TRDL vector system is linked with caveoli-mediated endocytosis we performed laser scanning fluorescent confocal microscopic analysis by immune-staining the cells for caveolin-1 (blue) and TR (red).
The cells were not permeabilized to detect only the TR localized at the membrane. In our experiments we found a significant co-localization between caveolin and TR (pink colour). The fluorescent signal of TR was visualized at different time points during transfection (Figure 16-A: a to c). The signal was initially localized on the cell membrane. However, a time dependant clearance of the signal from the cell membrane was observed.
In order to show the time dependent cellular internalization of TR, the cells were permeabilized and then stained with streptavidin-Cy3 (red) for intracellular tracking of TR. We observed a strong Cy3 signal at the cell periphery at 2 h, which gradually moved into the cell cytoplasm after longer incubations resulting in a strong cytoplasmic signal after 15 h (Figure 16-A: d to f).
In order to examine if DNA internalization corresponded with that of TR, the cells were transfected with a 400 bp Fam-labeled DNA fragment using TR and the transfection process was examined by visualizing the uptake of Fam-labeled DNA into the cells at different time points (Figure 16-B: a to c). We employed Cy3-tagged secondary antibody for caveolin-1 in these experiments and labeled the cytoplasmic and nuclear compartments of the cell with WGA-633 and TO-PRO-3 for the membrane and nucleus, respectively. We observed a strong correlation in time between the cellular internalization of DNA and that of TR.
Figure 16: TatRGD co-localization with Cav-1 and its cellular internalization (A). A549 cells were transfected with 400bp DNA fragment using TRDL vectors, fixed at 2 h a), 4 h (A-b) and 15 h (A-c) time points and immuno-stained with anti-Cav-1-Cy-5 (blue) and
streptavidin-20 µm
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A-a)
A-b )
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Cav-1 Tat-RGD Merge A-d )
A-e)
A-f)
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Cy-3 (red) for caveolins and biotin-TR respectively. We observed co-localization (merge) of TR with Cav-1 (pink) and a time dependant clearance of TR from the cell membrane. When cells were permeabilized and stained with streptavidin-Cy-3, we observed a time dependant internalization of TR (red) into the cytoplasmic compartment of the cells (A-d to f). TatRGD mediated DNA internalization in cellular compartments (B). A549 cells were transfected with 400bp Fam labeled DNA fragment using TRDL vectors, fixed at 2 h a), 4 h b) and 15 h (B-c) time points and immuno-stained with anti-Cav-1-Cy-3 (red) for Caveolins. In order to distinguish the cellular compartments, the cells were stained with WGA-633 (blue) and TO-PRO-3 (blue) for cell membrane and nucleus respectively. We observed a time dependant cellular internalization of Fam labeled DNA, which corresponded with the internalization patterns of TR in permeabilized cells.
Cellular internalization of TRDL in relation with caveolin expression:
Pulmonary cell-line (A549) and primary cells (SMCPA) were transfected with Fam labeled DNA fragment and the cells were immuno-stained for Cav-1 (with secondary antibody conjugated with Cy-3). We observed a strong correlation between the DNA (Fam) internalization and the expression pattern of caveolins in these cell types. A549 cell-line exhibited strong membrane caveolin staining and also higher Fam internalization (figure 17-A). While SMCPA primary cells exhibited weak membrane caveolin staining and also lower Fam internalization (figure 17-B)
A549 cell-line
SMCPA primary cells A
B
Figure 17: Cellular internalization of TRDL in relation with caveolin expression. A549 cell-line SMCPA primary cells were transfected with Fam-DNA (green) employing TRDL vectors and stained with WGA-633 (blue), TO-PRO-3 (blue), anti-Cav-1-Cy-3 (red) for cell-membrane, nucleus and caveolins respectively. We observed that, the caveolin staining at the cell membrane was strong in A549 cells (A: above) and relatively weak in SMCPA cells (B: above).
We observed that, Fam-DNA localization in the cellular and nuclear compartments was strong in A549 cells (A: below) and relatively weak in SMCPA cells (B: below).
3.1.9 Functional assay:
Prevention of Caveoli formation by cholesterol depletion:
To demonstrate further that internalization of TRDL complexes exploit caveolar endocytic system, we tested the effect of drugs on TRDL mediated internalization.
Methyl-β-cyclodextrin specifically disrupts the formation of caveoli with out interfering with other modes of endocytosis (75). Cells were pretreated with 1% methyl-β-cyclodextrin for 30 m and transfected with TRDL complexes. Prevention of caveoli formation drastically decreased the transfection efficiency with TRDL vector system while that with the standard vector system however remained unaffected (figure 18), suggesting the role of caveoli in the cellular internalization of TRDL complexes.
Figure 18: Prevention of Caveoli formation by cholesterol depletion. A549 Cells were pre-treated with 1% MBC for 30 m and transfected with TRDL and DL vector systems and the luciferase reporter gene expression was measured. Cells untreated with MBC were taken as controls for both TRDL and DL transfections.
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